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Title: Glycomic analysis of biomedically important O-glycoconjugates
Author: Ismail, Mohd Nazri
ISNI:       0000 0004 2697 0742
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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It has been established that all cells carry an array of glycans attached to proteins and lipids that are crucial in the interaction between cells and the surrounding matrix. Proteins are mainly glycosylated on asparagines (N-glycosylation) and serine or threonine residues (O-glycosylation). Compared to N-glycans, O-glycans offer a higher degree of structural ambiguity due to the existence of several types and cores. This is believed to contribute to the relative lack of knowledge on these molecules. Therefore, improvement to the current methodologies of structural studies is a prerequisite to complement the immense functional findings of O-glycoconjugates in biological systems. This thesis discusses the structural characterisation, regulation and biological roles of O-glycans. The overall aim was to optimise O-glycomic mass spectrometric analysis to help illuminate the phenotypic findings from our collaborators in three separate but related projects. The methodologies utilised involving MALDI-TOF/TOF-MS, GC-EI-MS, ESI-QTOF-MS and MALDI-QIT-TOF-MS. The first project investigated the effects of core 2 GlcNAc transferase (C2GnT) deficiency in mice. This enzyme exists in three isoforms which are expressed differently in different tissues. Analysis of the single knockout of each of these isoenzymes as well as the triple knockouts has allowed the investigation of their unique and overlapping functions. The outcomes of this study include characterisation of alterations of not just mucin-type O-glycans but also O-mannose glycan, which could be associated with several organ lesions and system failures. The second project focused on the gastric mucosa of mice with deficiency in α1,2-fucosyltranferase (FuT2). This enzyme plays an important role in decorating the mucosal mucins with ABH-blood group and Lewis antigens that are known to interact with various gut flora including the pathogen Helicobacter pylori. It has been shown that the binding of H. pylori via BabA adhesins was significantly impaired with the loss of H antigens and Lewis y on O-glycans. The third project investigated the regulation of mucin-type O-glycosylation. The protein Src has been recognised to play an essential role in the localisation of ppGalNAc transferases, the initiating enzyme of O-glycosylation, in the endoplasmic reticulum and Golgi apparatus. Therefore, it could be inferred that Src influences the regulation of protein O-glycosylation. The NIH3T3 and NBT-II cell lines with different levels of Src or different localisation of ppGalNAcT-2 have been analysed in order to identify the changes on the structures of O-glycans and the relative abundances of cores 1 and 2. Valuable information has been gathered which could lead to further investigative work to better understand the role of Src in the regulation of protein O-glycosylation.
Supervisor: Dell, Anne ; Haslam, Stuart Sponsor: Malaysian Ministry of Higher Education ; Universiti Sains Malaysia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral